Ewing sarcoma is a common pediatric tumor arising in bone and soft tissue. At the molecular level, all Ewing tumors contain chromosomal translocations that create in-frame fusion genes encoding the N-terminus of the RNA binding protein EWS fused to the DNA-binding domain of one of several ETS proteins, most commonly FLI-1. Related EWS fusion proteins are found to five other types of human sarcoma. The exact mode of action of these chimeric proteins in tumorigenesis is unknown. While the transcription activity of EWS/FLI contributes significantly to transformation, it is not absolutely required. The goal of our work is to determine the mechanisms through which EWS chimeric transcription factors contribute to the genesis of Ewing sarcoma. Histologically, Ewing tumors are poorly differentiated and lack distinguishing morphological features. Consequently, the cell of origin of Ewing tumors remains unclear. Progress in this field has been limited by not knowing the correct cell background in which to study EWS/FLI activity. We hypothesized that the mesenchymal progenitor cell derived from bone marrow is a good candidate for the cell of origin. Our preliminary experiments demonstrated that EWS/FLI alters the morphology and blocks the differentiation of these cells, features that are consistent with the undifferentiated appearance of Ewing tumors. We will determine the effect of EWS/FLI expression on the growth, differentiation, transformation potential, and changes in gene expression profiles, in this cellular background. The effects of cooperating mutations in the tumor suppressor genes INK/AIARF and p53, which are mutated in Ewing tumors, will be assessed, and we will identify novel genetic changes that enhance EWS/FLI-mediated transformation. Gene expression profiles and candidate cooperating genes defined in our in vitro system will be evaluated in human Ewing sarcoma samples to relate our experimental findings to the human disease. Finally, we will establish a mouse model to evaluate the consequences of EWS/FLI expression on tumorigenesis and development in vivo.